Cysteine proteases associated with human disease states can be grouped into three categories: (1) lysosomal cathepsins; (2) cytosolic calpains; and (3) procaryotic enzymes with autocatalytic activation. Cathepsins B, H, and L are cysteinyl proteases involved in normal protein degradation. As such, they are generally located in the lysosomes of cells. When these enzymes are found extralysosomaly they have been implicated by use of synthetic substrate technology and by natural endogenous inhibitors as playing a causative role in a number of disease states such as rheumatoid arthritis, osteo arthritis, pneumocystis carinii, schistosomiasis, trypanosoma cruzi, trypanosoma brucei, brucei, Crithidia fusiculata, malaria, periodontal disease, tumor metastasis, metachromatic leukodystrophy, muscular dystrophy, etc. For example, a connection between cathepsin B-type enzymes and rheumatoid arthritis has been suggested in van Noorden and Everts, "Selective Inhibition of Cysteine Proteinases by Z-Phe-Ala-CH.sub.2 F Suppresses Digestion of Collagen by Fibroblasts and Osteoclasts," 178 Biochemical and Biophysical Research Communications 178; Rifkin, Vernillo, Kleekner, Auszmann, Rosenberg and Zimmerman, "Cathepsin B and L Activities in Isolated Osteoclasts," 179 Biochemical and Biophysical Research Communications 63; Grinde, "The Thiol Proteinase Inhibitors, Z-Phe-Phe-CHN.sub.2 and Z-Phe-Ala-CHN.sub.2, Inhibit Lysosomal Protein Degradation in Isolated Rat Hepatocytes," 757 Biochimica et Biophysica Acta 15; Mason, Bartholomew and Hardwick, "The Use of Benzyloxycarbonyl[.sup.125 I]iodotyrosylalanyldiazomethane as a Probe for Active Cysteine Proteinases in Human Tissues," 263 Biochem. J. 945; van Noorden, Smith and Rasnick, "Cysteine Proteinase Activity in Arthritic Rat Knee Joints and the Effects of a Selective Systemic Inhibitor, Z-Phe-Ala-CH.sub.2 F," 15 J. Rheumatol. 1525; and van Noorden, Vogels and Smith, "Localization and Cytophotometric Analysis of Cathepsin B Activity in Unfixed and Undecalified Cryostat Sections of Whole Rat Knee Joints," 37 J. Histochemistry and Cytochemistry 617. A connection between cathepsin B and osteo arthritis has been suggested in Delaisse, Eeckhout and Vaes, "In Vivo and In Vitro Evidence for the Involvement of Cysteine Proteinases in Bone Resorption," 125 Biochemical and Biophysical Research Communications 441; a connection between cathepsin B and pneumocystis carinii has been suggested in Hayes, Stubberfield, McBride and Wilson, "Alterations in Cysteine Proteinase Content of Rat Lung Associated with Development of Pneumocystis Carinii Infection," 59 Infection and Immunity 3581; a connection between cysteine proteinases and schistosomiasis has been suggested in Cohen, Gregoret, Amiri, Aldape, Railey and McKerrow, "Arresting Tissue Invasion of a Parasite by Protease Inhibitors Chosen With the Aid of Computer Modeling," 30 Biochemistry 11221. A connection between cysteine proteinases and trypanosoma cruzi, trypanosoma brucei brucei and crithidia fasciculata has been suggested in Ashall, Harris, Roberts, Healy and Shaw, "Substrate Specificity and Inhibitor Sensitivity of a Trypanosomatid Alkaline Peptidase," 1035 Biochimica et Biophysica Acta 293, and/or in Ashall, Angliker and Shaw, "Lysis of Trypanosomes by Peptidyl Fluoromethyl Ketones," 170 Biochemical and Biophysical Research Communications 923. A connection between cysteine proteinases and malaria has been suggested in Rosenthal, Wollish, Palmer and Rasnick, "Antimalarial Effects of Peptide Inhibitors of a Plasmodium Falciparum Cysteine Proteinase," 88 J. Clin, Invest. 1467, and in Rosenthal, Lee and Smith, "Inhibition of a Plasmodium Vinckei Cysteine Proteinase Cures Murine Malaria," (in press). A connection between cathepsin B and tumor metathesis has been suggested in Smith, Rasnick, Burdick, Cho, Rose and Vahratian, "Visualization of Time-Dependent Inactivation of Human Tumor Cathepsin B Isozymes by a Peptidyl Fluoromethyl Ketone Using a Fluorescent Print Technique," 8 Anti-cancer Research 525. A connection between cathepsin B and cancer has been suggested in Gordon and Mourad, 2 Blood Coagulation and Fibrinolysis 735. A connection between cathepsin B and periodontal disease has been suggested in Cox, Cho, Eley and Smith, "A Simple, Combined Fluorogenic and Chromogenic Method for the Assay of Proteases in Gingival Crevicular Fluid," 25 J. Periodont. Res. 164; Uitto, Larjava, Heino and Sorsa, "A Protease of Bacteroides Gingivalis Degrades Cell Surface and Matrix Glycoproteins of Cultured Gingival Fibroblasts and Induces Secretion of Collagenase and Plasminogen Activator," 57 Infection and Immunity 213; Kunimatsu, Yamamoto, Ichimaru, Kato and Kato, "Cathepsins B, H and L Activities in Gingival Crevicular Fluid From Chronic Adult Periodontitis Patients and Experimental Gingivitis Subjects," 25 J Periodont Res 69; Beighton, Radford and Naylor, "Protease Activity in Gingival Crevicular Fluid From Discrete Periodontal Sites in Humans With Periodontitis or Gingivitis"; 35 Archs oral Biol. 329; Cox and Eley, "Preliminary Studies on Cysteine and Serine Proteinase Activities in Inflamed Human Gingiva Using Different 7-Amino-4-Trifluoromethyl Coumarin Substrates and Protease Inhibitors," 32 Archs oral Biol. 599; and Eisenhauer, Hutchinson, Javed and McDonald, "Identification of a Cathepsin B-Like Protease in the Crevicular Fluid of Gingivitis Patients,"62 J. Dent Res 917. A connection between cathepsin B and metachromatic leukodystrophy has been suggested in von Figura, Steckel, Conary, Hasilik and Shaw, "Heterogeneity in Late-Onset Metachromatic Leukodystrophy. Effect of Inhibitors of Cysteine Proteinases," 39 Am J Hum Genet. 371; a connection between cathepsin B and muscular leukodystrophy has been suggested in Valentine, Winand, Pradhan, Moise, de Lahunta, Kornegay and Cooper, "Canine X-Linked Muscular Dystrophy as an Animal Model of Duchenne Muscular Dystrophy: A Review," 42 Am J Hum Genet 352; a connection between cathepsin B and rhinovirus has been suggested in Knott, Orr, Montgomery, Sullivan and Weston, "The Expression and Purification of Human Rhinovirus Protease 3C," 182 Eur. J. Biochem, 547; a connection between cathepsin B and kidney disease has been suggested in Baricos, O'Connor, Cortez, Wu and Shah, "The Cysteine Proteinase Inhibitor, E-64, Reduces Proteinuria in an Experimental Model of Glomerulonephritis," 155 Biochemical and Biophysical Research Communications. 1318; and a connection between cathepsin B and multiple sclerosis has been suggested in Dahlman, Rutschmann, Kuehn and Reinauer, "Activation of the Multicatalytic Proteinase from Rat Skeletal Muscle by Fatty Acids or Sodium Dodecyl Sulphate," 228 Biochem. J. 171.
Connections between certain disease states and cathepsins H and C have also been established. For example, cathepsin H has been directly linked to the causative agents of Pneumocystis carinii and in the neuromuscular diseases Duchenne dystrophy, polymyositis, and neurogenic disorders. Stauber, Riggs and Schochet, "Fluorescent Protease Histochemistry in Neuromuscular Disease," Neurology 194 (Suppl. 1) March 1984; Stauber, Schochet, Riggs, Gutmann and Crosby, "Nemaline Rod Myopathy: Evidence for a Protease Deficiency," Neurology 34 (Suppl. 1) March 1984. Similarly, cathepsin C has been directly linked to muscular diseases such as hemaline myopathy, to viral infections, and to processing and activation of bone marrow serine proteases (elastase and granzyme A). McGuire, Lipsky and Thiele, "Generation of Active Myeloid and Lymphod Granule Serine Proteases Requires Processing by the Granule Thiol Protease Dipeptidyl Peptidase I, 268 J, Biol. Chem. 2458-67; L. Polgar, Mechanisms of Protease Action (1989); Brown, McGuire and Thiele, "Dipeptidyl Peptidase I is Enriched in Granules of In Vitro- and In Vivo-Activated Cytotoxic T Lymphocytes," 150 Immunology 4733-42. The Frown et al. study effectively demonstrated the feasibility of inhibiting cathepsin C (DPP-I) in the presence of other cysteinyl enzymes based on substrate specificity. Unfortunately, the diazoketones used in that study are believed to be mutagenic and not appropriate for in vivo application.
The cytosolic or membrane-bound cysteine proteases called calpains have also been implicated in a number of disease states. For example, calpain inhibitor can be useful for the treatment of muscular disease such as muscular dystrophy, amyotrophy or the like, 25 Taisha (Metabolism) 183 (1988); 10 J. Pharm. Dynamics 678 (1987); for the treatment of ischemic diseases such as cardiac infarction, stroke and the like, 312 New Eng. J. Med. 159 (1985); 43 Salshin Igaku 783 (1988); 36 Arzneimittel Forschung/Drug Research 190, 671 (1986); 526 Brain Research 177 (1990); for improving the consciousness disturbance or motor disturbance caused by brain trauma, 16 Neurochemical Research 483 (1991); 65 J. Neurosurgery 92 (1986); for the treatment of diseases caused by the demyelination of neurocytes such as multiple sclerosis, peripheral nervous neuropathy and the like, 47 J. Neuochemistry 1007 (1986); and for the treatment of cataracts, 28 Investigative Ophthalmology & Visual Science 1702 (1987); 34 Experimental Eye Research 413 (1982); 6 Lens and Eye Toxicity Research 725 (1989): 32 Investigative Ophthalmology & Visual Science 533 (1991).
Calpain inhibitors may also be used as therapeutic agents for fulminant hepatitis, as inhibitors against aggregation of platelet caused by thrombin, 57 Thrombosis Research 847 (1990); and as a therapeutic agent for diseases such as breast carcinoma, prostatic carcinoma or prostatomegaly, which are suspected of being caused by an abnormal activation of the sex hormone receptors.
Certain protease inhibitors have also been associated with Alzheimer's disease. See. e.g., 11 Scientific American 40 (1991). Further, thiol protease inhibitors are believed to be useful as anti-inflammatory drugs, 263 J. Biological Chem. 1915 (1988); 98 J. Biochem. 87 (1985); as antiallergic drugs, 42 J. Antibiotics 1362 (1989); and to prevent the metastasis of cancer, 57 Seikagaku 1202 (1985); --Tumor Progression and Markers 47 (1982); and 256 J. Biological Chemistry 8536 (1984).
Further, cysteine protease has been shown to process the convertase enzyme of interleukin-1, a cytokine that is implicated in septic shock, wound healing, and the growth of certain leukemias. See, e.g., Thornberry et al., 356 Nature 755; Goll et al., 74 Biochimi 225 (1992), and Bioworld Today, Vol. 3, No. 67, p. 1, Apr. 3, 1992.
Although a number of cysteine proteinase inhibitors have been identified, most of these have drawbacks for in vivo use. In particular, drawbacks such as reversibility of inhibition, lack of specificity, and rapid clearance from the body have been associated with prior art inhibitors. The microbial products antipain and leupeptin, for example, are effective but reversible inhibitors of cysteine proteinase (McConnell et al., 33 J. Med, Chem. 86-93; Sutherland et al. 110 Biochem. Biophys. Res. Commun, 332-38), and also inhibit certain serine proteinases (Umezawa, 45 Meth. Enzymol 678-95). The compound E64 and its synthetic analogues are more selective inhibitors (see, e.g., Barret et al., 201 Biochem. J. 189-98, and Grinde, 701 Biochem. Biophys. Acta. 328-33), but disappear too quickly from the circulation for in vivo use (Hashida et al. 91 J, Biochem. 1373-80).
To date, two classes of peptidyl ketone inhibitors have been identified. One class, originally described by Abeles, comprises reversible inhibitors of both serine and cysteine proteases. For example, Abeles et al. describe trifluoromethyl ketones of the form: ##STR1## Abeles et al., "Fluoroketone Inhibitors of Hydrolytic Enzymes," 24 Biochemistry 1813-17, (1985).
Similarly, reversible inhibitors to serine proteases also exist in their hydrated form: ##STR2## and therefore become transition state analog inhibitors. Further, Hu et al., "Inhibition of Cathepsin B and Papain by Peptidyl .alpha.-ketoEsters, .alpha.-ketoAmides, .alpha.-Diketones, and .alpha.-ketoAcids", 281 Archives of Biochemistry and Biophysics 271-274 (1990) describe compounds of similar reactivity of the form:
Most recently, European Patent publication EP0-525420-Al disclosed peptidyl ketones that were activated by the permanent (nonfissionable) placement of methylthiomethyl ring or amine, thus making reversible inhibitors of reduced efficacy.
The difference between these noncleavable reversible inhibitors and the irreversible inhibitors of the present invention is that the former inhibitors remain intact in the active site constantly partitioning with the external medium while the latter cleaves and leaves the peptide portion irreversibly bonded to the enzymes active site, thereby permanently disabling the enzyme. This latter mechanism translates into lower required doses of therapeutic agent.
The most promising type of cysteine proteinase inhibitors have an activated carbonyl with a suitable .alpha.-leaving group fused to a programmed peptide sequence that specifically directs the inhibitor to the active site of the targeted enzyme. Once inside the active site, the inhibitor carbonyl is attacked by a cysteine thiolate anion to give the resulting hemiacetal, which collapse3 via a 1,2-thermal migration of the thiolate and subsequent displacement of the .alpha.-keto-leaving group. The bond between enzyme and inhibitor is then permanent and the enzyme is irreversibly inactivated.
The usefulness of an inhibitor in inactivating a particular enzyme therefore depends not only on the "lock and key" fit of the peptide portion, but also on the reactivity of the bond holding the .alpha.-leaving group to the rest of the inhibitor. It is important that the leaving group be reactive only to the intramolecular displacement via a 1,2-migration of sulfur in the breakdown of the hemithioacetal intermediate.
Groundbreaking work regarding cysteine proteinase inhibitors having an activated carbonyl, a suitable .alpha.-leaving group and a peptide sequence that effectively and specifically directs the inhibitor to the active site of the targeted enzyme was disclosed in U.S. Pat. No. 4,518,528 to Rasnick, incorporated herein by reference. That patent established peptidyl fluoromethyl ketones to be unprecedented inhibitors of cysteine proteinase in selectivity and effectiveness. The fluoromethyl ketones described and synthesized by Rasnick included those of the formula: ##STR3## wherein R.sub.1 and R.sub.2 are independently selected from the group hydrogen, alkyl of 1-6 carbons, substituted alkyl of 1-6 carbons, aryl, and alkylaryl where the alkyl group is of 1-4 carbons; n is an integer from 1-4 inclusive; X is a peptide end-blocking group; and Y is an amino acid or peptide chain of from 1-6 amino acids.
Peptidylketone inhibitors using a phenol leaving group are similar to the peptidyl fluoroketones. As is known in the art, oxygen most closely approaches fluorine in size and electronegativity. Further, when oxygen is bonded to an aromatic ring these values of electronegativity become even closer due to the electron withdrawing effect of the sp.sup.2 carbons. The inductive effect of an .alpha.-ketophenol versus an .alpha.-ketofluoride when measured by the pKa of the .alpha.-hydrogen, appears comparable within experimental error.
Unfortunately, the leaving groups of prior art cysteine protease inhibitors have presented problems of toxicity, solubility, etc. For example, the inhibitors disclosed by Krantz et al. in U.S. Pat. No. 5,055,451 have been found to be unacceptably toxic when introduced into animals such as rabbits or dogs.
A need therefore exists for cysteine protease inhibitors with improved solubility and toxicity profiles, and which are particularly suitable for in vivo use. The present invention addresses that need.